Frequently Asked Questions

You can find answers for many of your general questions about the PG1639, PG1075 and PG0430 Detection Kit below. For more specific requests and questions, please contact our Technical Support for further assistance.

How should I store the kit?
These Detection Kits should always be kept at -20°C in the dark and thawed on the ice while using.

Can I use blood collection tubes containing Heparin as anticoagulant?
It is highly recommended to use blood collection tubes containing either sodium citrate or EDTA as the anticoagulants. Heparin may have an inhibitory effect on PCR.

What is the required quality of gDNA?
The OD260/280 ratio of gDNA should be between 1.7 and 2.0.

What is the appropriate amount of DNA in the reaction?
The appropriate amount of gDNA should be between 25-100 ng/reaction.

How many reactions does each sample need?
Only one independent PCR reactions should be conducted simultaneously for each DNA sample. Four control reactions (three for positives and one for negative) should be companied on each run.

What dyes do these kits use?
FAM and VIC fluorescence.

What is the detection method of these Detection Kits?
This is one kind of SNP genotyping methods, and we use two specific probes labeled with different fluorescence to detect a SNP site.

How do I determine the genotype of sample?
You can determine the genotype of sample the same as the positive control nearest to it in the Allelic Discrimination map.

Can I use Post Read function only?
You can perform the test without Pre Read. However, It is strongly recommended to use Pre Read to avoid the interference from background noise if your instrument has this function.

Should Positive controls have fluorescent signal?
Positive controls should have strong fluorescent signals and can show four obvious regions with NTC on Allelic Discrimination map.

Should each sample have fluorescent signal of SNP?
Each sample has its own SNP types which should have fluorescent signal. If there are some interferents in samples, the locations of signal may move toward the origin (NTC site).

Do I need to duplicate my tests?
Based on the description on package insert, duplication is not always needed. It needs to be conducted according to the QC guidance of each lab.

Does the NTC show obvious fluorescent signal?
NTC is a control without DNA sample, and it may still have very weak signal, which has no effect on Allelic Discrimination. High fluorescent signal shown represents an artificial mistake and has sample DNA contamination(s).

How do I set up the software program of the quantitative PCR instrument?
PharmiGene provides program setting instructions for a few quantitative PCR detection systems. Please do not hesitate to contact us for the further information if needed.

How do I contact the Technical Support dpartment of PharmiGene?
Please call +886-2-26959800 or email service@pharmigene.com to request a PGI's service.

Troubleshooting Guide

This trouble shooting guideline may be helpful in solving your problems during analyzing. The procedures of performing PG1639, PG1075 and PG0430 Detection Kits include genomic DNA extraction, real-time PCR amplification, and data analysis. We include the most frequently problems and their solutions. If the problems still exist, please contact us for further helping. The scientists in Pharmigene Technical Support are always happy to answer your questions to help you perform the PG1639, PG1075 and PG0430 Detection Kits successfully.

Proublems

Possible Cause(s)

Recommendation(s)

a.

No amplification can be monitored

Wrong channel has been chosen.

-If the quantitative PCR instrument you use which automatically detects fluorescence intensities of all the channels, you can simply reset the right plate information and channel (FAM/VIC) to recalculate data.
-If the instrument does not automatically collect whole channels, you need to abort and redo the run.

Pipetting errors or omitted reagents.

-Check the set-up of the reaction and missing reagents.
-Redo the PCR run.
-Always run a positive control along with samples.

Wrong program.

Check the amplification program which is followed as the package insert.

Instrument broken down.

Contact the technical support of instrument's company.

b.

Weaker fluorescent signal

Using Heparin as the anticoagulant in blood collection.

-Use either sodium citrate or EDTA as the anticoagulants in blood collection.
-Extract DNA within 3 days after blood withdrawing.

A total gDNA of 25~100 ng/reaction is appropriate for these Detection Kits.

Poor gDNA quality

-The OD260/280 ratio between 1.7 and 2.0 is required for performing these Detection Kits.
-Clean up samples by a purification kit or re-extract DNA from blood.
-Qiagen QIAamp® DNA Blood Mini Kit is recommended for DNA extraction.

Very low starting amount of DNA

-Qiagen QIAamp® DNA Blood Mini Kit is recommended for DNA extraction.
-A total gDNA of 25~100 ng/reaction is appropriate for these Detection Kits.

Freeze and thaw reagents more than three cycles

Aliquot and freeze kit reagents to ensure the kit quality and real-time PCR performance. (Based on Pharmigene stability test, these Detection Kits can be freeze and thaw for three cycles and perform normally.)

Reagents are kept in inappropriate temperature

-Keep kits in -20°C for long-term storage.
-Keep the reagents on ice when thaw them.

-The OD260/280 ratio between 1.7 and 2.0 is required for performing these Detection Kits.
-Clean up samples by a purification kit or re-extract DNA from blood.
-Qiagen QIAamp® DNA Blood Mini Kit is recommended for DNA extraction.

Very low starting amount of DNA

-Qiagen QIAamp® DNA Blood Mini Kit is recommended for DNA extraction.
-A total gDNA of 25~100 ng/reaction is appropriate for these Detection Kits.

Components are not homogeneously mixed

Mix components completely.

Contaminations

-Repeat the run.
-Always wear gloves when preparing the reactions.

Reaction solution is not in the bottom of tube.

Leave reaction solution in the bottom of tube by higher centrifugal speed.